1. Field of the Invention
This invention relates to methods for determining the distance between particles and a surface to which the particles are adhered. Particles are labeled with a detectable label and adhered to a surface which may be a natural surface or an artificial surface. Label is detected from these particles under different conditions of adherence and a value is determined that reflects the strength of adherence. These methods are useful for determining the physical nature of a surface of any particle and surface phenomenon such as events associated with the cell adhesion, cell spreading and ligand-receptor interactions. These methods are so used for detecting and analyzing cell-associated pathological conditions and the physical nature of any surface.
2. Description of the Background
Cell adhesion to other cells and to biological surfaces is required for normal physiological processes such as leukocyte trafficking and the immune response, and is also an important factor in the development of pathological conditions associated with ischemia-reperfusion damage, autoimmune diseases and tumor metastasis. One of the principal cells in the adhesive process is the leukocyte. Adhesion of leukocytes involves specific interactions between complementary adhesion molecules present on both the leukocyte and endothelial surfaces. Subsets of leukocytes have different sets of adhesion receptors and each subset has different affinities and abilities to spread along endothelial cells (M. B. Lawrence and T. A. Springer, Cell 65:859-73, 1991; Y. Shimizu et al., Immunol. Today 13:106-12, 1992).
Stimulation of the inflammatory process is closely associated with control of the adhesive properties of leukocytes, endothelial cells and other cells. Cellular adhesion can be initiated or enhanced by inflammatory agents and other chemical inducers. For example, neutrophils can be activated with Fmet-leu-phe or phorbol esters such as phorbol myristic acetate (PMA). These agents and other chemical and biological inducers cause increased expression of adhesion molecules on cell surfaces, and potentiate adhesion, cell spreading and transendothelial migration (M. A. Arnaout et al., J. Cell. Physiol. 137:305-09, 1988; S. S. Smyth et al., Blood 81:2827-43, 1993). Activation of endothelial cells with IL-1, LPS or TNF produces increased expression of the endothelial cell adhesion ligands ICAM-1, VCAM-1 and ELAM-1, which also mediate strong adhesion of leukocytes (F. W. Luscinskas et al., J. Immunol. 146:1617-25, 1991; B. C. Hakket et al., Blood 78:2721-26, 1991).
Another particularly good example of adhesion is margination, the deposition of white cells within microvessels. As white cells tumble increasingly slowly along capillary and venule walls, the cells eventually come to rest and adhere to vessel endothelium. Damage can occur when the endothelium becomes lined with these cells. This phenomenon, not surprisingly, is referred to as pavementing and may be related to the development of arteriosclerosis and associated diseases.
Cells in culture mimic many types of adhesive behaviors and form specialized contacts between cells and with the extracellular matrix (ECM). These surface-to-surface interactions are believed to be identical to those formed in vivo (B. Geiger et al., J. Mol. Biol. 101:1523-31, 1985). Inflammatory mediators such as C3a, FMLP and LPS specifically stimulate increased expression and increased affinity of many of these proteins. Conversely, other inflammatory mediators inhibit adhesion and may serve to terminate inflammatory responses. The overall importance of these adhesion molecules is highlighted by a genetic deficiency, referred to as leukocyte adhesion deficiency, in which there is a deficiency in the .beta. and sometimes the .alpha. subunit. Patients with this disorder suffer from increased and recurrent bacterial and vital infections.
Many different families of proteins are important in cell-to-cell and cell-to-surface interactions. One group of adhesion molecules found on leukocytes is called the .beta..sub.2 -integrins and consists of a family of three glycoproteins LFA-1, MO-1 and p150/95. LFA-1, for example, participates in strong leukocyte-endothelial interactions, mediating firm adhesion, spreading and transendothelial migration (M. B. Lawrence and T. A. Springer, Cell 65:859-73, 1991; E. C. Butcher, Cell 67:1033-36, 1991). Structurally, these proteins are heterodimer complexes comprised of identical .beta. (beta) subunits and different .alpha. (alpha) subunits with a molecular weight range of 150,000 to 180,000 KDa.
Within this well studied family of integrins there are also specific cell surface proteins that mediate biological events involving cell-matrix and cell-cell interactions. The integrins provide a functional linkage between the extracellular matrix and the cells' interior providing a mechanism to effect cellular responsiveness to the extracellular environment. Some integrins also bind, and with great specificity, to a host of different types of ligands. Specificity is conferred by the subunit composition of the integrin complex. At least three major subgroups can be distinguished by the .beta. subgroup and six by the .alpha. subgroup. These protein complexes bind to extracellular ligands such as laminin, collagen and fibronectin, and to cytoskeletal proteins such as vinculin, talin and .alpha.-actinin, via their cytoplasmic tails.
Integrins are an essential requirement for leukocyte adhesion and are also involved with functional activation of many cell types. Cellular adhesion requires a number of specific interactions which occur at the cell surface including podosomes, point contacts, dot contacts, focal contacts which may be uniformly distributed across the cell surface or localized at specific sites. Podosomes are large aggregates, on the order of 200-400 nm, which contain actin. Point contacts and dot contacts are smaller than podosomes, about 90-200 nm, and are closely apposed to the substratum of the cell. Focal contacts, or adhesion plaques are structures which traverse the plasma membrane linking the extracellular substrate with components of the cytoskeleton and contain concentrated amounts of integrin (K. Burridge et al., Ann. Rev. Cell Biol. 4:487-525, 1988). The ability of a cell to form focal contact correlates with its ability to assemble an organized cytoskeleton, a prerequisite to cell spreading and migration (E. Dejana et al., J. Cell Biol. 107:1215-23, 1987).
The early stages of cell attachment and spreading are believed mediated exclusively by integrins in point contacts. Unlike focal contacts, point contacts contain clathrin and rarely distribute with actin or vinculin. It has been proposed that different classes of adhesion molecules mediate the progression of weak point contacts to strong adhesion which results in spreading (E. C. Butcher, Cell 67:1033-36, 1991). In addition, strong adhesion and flattening or spreading is required prior to transendothelial migration of leukocytes (F. W. Luscinskas et al., J. Immunol. 146:1617-25, 1991; B. C. Hakket et al., Blood 78:2721-26, 1991). For example, the CD18 (cluster differentiation type 18) integrins, LFA-1 and MO-1, are thought to mediate strong adhesion, and antibodies against these integrins block strong adhesion, spreading and transendothelial migration.
Cellular adhesion is one of the many systems in which the presence of specific molecules is required for adhesion to occur (W. A. Frazier et al., Cellular Recognition, Alan R. Liss, Inc., New York, 1982). In both cell-to-cell and cell-to-surface adhesion, bridges are formed by adhesion molecules such as integrins and it appears clear that a substantial number of bridges must be formed to achieve successful adhesion. This later requirement indicates that there exists a repulsive barrier which must be overcome to permit adhesion to occur. The existence of a repulsive barrier is further supported by studies which show that even when numerous bridging molecules are present, cells must frequently be forced into close contact by centrifugation before strong bonding occurs.
There are many possibilities inherent in thermodynamic adhesion models. Each model can explain the thermodynamic considerations for a small number of cell surface interactions. None have been able to explain every type of interaction, however, a few general observations can be made.
First, specific bridging molecules, or receptors, become concentrated in regions of cell-cell or cell-substrate contact. Such accumulations of receptors in contact areas have been observed and can be deduced from the forces required to disrupt aggregates. Accumulations of receptors serve as transduction mechanisms for triggering cellular responses. This is demonstrated in the immune system in which activation, phagocytosis and exocytotic granular release have each been demonstrated to be controlled by contact. Redistribution of receptors could also serve as a signal for polarization of the cell membrane relative to the site of adhesion. Internal cell structural elements such as cytoskeletal elements, may be able to sense this polarization. If so the interior of the cell can also be polarized. Specialized cell-cell junctions such as synapses, tight junctions and gap junctions, could be the natural consequence of receptor accumulation in regions of cell-cell contact. Details of the junction to be affected are determined by specificities of the receptors and their interactions with each other and with other molecules of the cell.
Another observation is that there are many phase transitions in cell adhesion. The existence of these transitions makes it clear that adhesion cannot be thought of according to only the basic laws of mass action. Contact between cells can only be stabilized by highly cooperative rearrangements of the interval variables of the cells.
Theoretical models of cell-cell interaction predict that the distance between two adherent cells in the area of contact is determined by a balance between nonspecific repulsion of hydrophilic polymers associated with the cell surface, and attraction through specific ligand-receptor bridges (G. I. Bell et al., Biophys. J. 45:1051-64, 1984). The physical nature of cell-cell, ligand-receptor bridges depends on many parameters, such as the type of the receptors, the density and direction of a stress force, blockage of the receptors with monoclonal antibodies or transfer of adhesion mediators to different ligand-receptor pairs. The process of adhesion itself may represent a regulatable continuum with attachment being mediated by a single small contact point between an adhering cell membrane and a surface of adhesion, which progresses to a flattening or spreading of the adhering cell along the surface of adhesion, fostering multiple points of contact between the cell and the surface.
Adhesion is more than a simple sum of the independent contributions of the various attractive forces, but a thermodynamic interaction of the attractive and repulsive forces causing the overall free energy of the interaction to favor one configuration over many other possible configurations. Unfavorable configurations have a relatively strong repulsive barrier and a weak, if any, attractive force. Favorable configurations have, in addition to a counterbalanced repulsive barrier, a compilation of attractive forces which achieve a successful degree of bridging.
Direct correlations between adhesive properties and pathological sequelae has been demonstrated for a number of diseases and disorders. For example, tumor metastasis is a complex process involving dynamic interactions between tumor cells and extracellular structures. Stages of malignancy have been demonstrated in sublines of an epithelial cell strain using differences in adhesion to tissue culture plastic (J. G. Steele et al., J. Cell Sci. 100:195-203, 1991). Andre et al. compared ten human melanoma cell lines and identified independent measurable parameters which correlated adhesion with characteristics of transformed cells (Cell. Biophys. 17:163-80, 1990). Boxberger and Paweletz related the influence of various substrata on rat tumor cell morphology, motility and invasiveness (Anticancer Res. 10:741-51, 1990). Recently, Repesh et at. demonstrated that adriamycin-induced inhibition of melanoma cell invasiveness is correlated with decreases in tumor cell motility and increases in focal contact formation (Clin. Exp. Metastasis 11:91-102, 1993).
Connections between adhesion and disease are not limited to neoplasias. A specific inheritable defect in neutrophil motility has been traced to abnormalities in a 47 KDa and a 89 KDa proteins of the cytoskeleton (T. D. Coates et ah, Blood 78:1338-46, 1991). Patients with this defect have increased and recurrent infections. Platelet activation and abnormal adhesion has been shown to be involved with atherosclerosis (S. K. Peng et al., Artery 20:122-34, 1993). Cellular adhesion has been implicated in muscular dystrophy (J. Cell Sci. 97:149-56, 1990), demyelination-related disorders (Ann. New York Acad. Sci. 605:1-14, 1990), and prostatitis (Urol. Int. 46:15-17, 1991). Furthermore, cellular adhesive properties have obvious importance in cell attachments to implants such as osteoclast attachments to a dental matrix (C. Wedenberg and S. Yumita, Endod. Dent. Traumatol. 6:255-59, 1990) and the formation of focal contacts by osteoblasts on orthopedic biomaterials (D. A. Puleo and R. Bizios, J. Biomed. Mater. Res. 26:291-301, 1992).
The development of reagents designed to prevent cell adhesion and pathological sequelae is being actively pursued for many of these pathological conditions. Anti-integrin antibodies have been identified that stimulate specific integrin function (K. M. Neugebauer and L. F. Reichardt, Nature 350:68-71, 1991; B. M. C. Chan and M. E. Hemlet, J. Cell Biol. 120:537-43, 1993). Whether this has more to do with increased expression or increased activation is not dear, but what is clear is that there are many agents which can effect, either directly or indirectly, the process of adhesion. Key features of this process, and certain pathological consequences, include adhesive interactions between minor cells and extracellular matrix components and enzymatic degradation of components. Properties such as invasiveness, growth, differentiation and motility are dependant to some degree on the adhesive properties of the tumor cells (L. A. Liotta et al., Cell 64:327-36, 1991). These properties have been ignored as a diagnostic means because there has been no accurate, reproducible and inexpensive method to measure cell surface distances.